Potassium Permanganate Probing of Pol II Open Complexes

INTRODUCTION

One measure of an activator’s ability to stimulate preinitiation complex assembly is stimulation of the RNA polymerase II (Pol II) open complex. The reagent most commonly used to visualize open complexes is potassium permanganate (KMnO₄). KMnO₄ modifies thymine residues in single-stranded DNA but not in double-stranded DNA. The modified thymines are detected by primer extension via linear polymerase chain reaction (PCR). In the PCR, Taq polymerase stalls at the modified thymines on the template. Reactions are performed at 30°C and contain DNA template (typically, 10-50 ng present on a 4- to 5-kbp plasmid), nuclear extract, activator, and nonspecific carrier DNA. DNA melting by Pol II in vitro is energy dependent, so ATP is added to the reaction mix. After a brief 30-min incubation to permit assembly of the preinitiation complex, permanganate is added and the DNA is modified for a period of 1 min or more. The DNA is then purified and subjected to primer extension analysis; the products are fractionated as a series of bands on polyacrylamide/urea sequencing gels. Reactions with and without activator are compared, as are reactions with and without ATP. Titrations of the DNA template and activator and a time course are recommended.